Targeted integration by homologous recombination enablesin situtagging and replacement of genes in the marine microeukaryoteDiplonema papillatum

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60076658%3A12310%2F20%3A43901331" target="_blank" >RIV/60076658:12310/20:43901331 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/60077344:_____/20:00537342

Výsledek na webu

<a href="https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.15130" target="_blank" >https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.15130</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1111/1462-2920.15130" target="_blank" >10.1111/1462-2920.15130</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Targeted integration by homologous recombination enablesin situtagging and replacement of genes in the marine microeukaryoteDiplonema papillatum

Popis výsledku v původním jazyce

Diplonemids are a group of highly diverse and abundant marine microeukaryotes that belong to the phylum Euglenozoa and form a sister clade to the well-studied, mostly parasitic kinetoplastids. Very little is known about the biology of diplonemids, as few species have been formally described and just one,Diplonema papillatum, has been studied to a decent extent at the molecular level. Following up on our previous results showing stable but random integration of delivered extraneous DNA, we demonstrate here homologous recombination inD. papillatum. Targeting various constructs to the intended position in the nuclear genome was successful when 5 &apos; and 3 &apos; homologous regions longer than 1 kbp were used, achieving N-terminal tagging with mCherry and gene replacement of alpha- and beta-tubulins. For more convenient genetic manipulation, we designed a modular plasmid, pDP002, which bears a protein-A tag and used it to generate and express a C-terminally tagged mitoribosomal protein. Lastly, we developed an improved transformation protocol for broader applicability across laboratories. Our robust methodology allows the replacement, integration as well as endogenous tagging ofD. papillatumgenes, thus opening the door to functional studies in this species and establishing a basic toolkit for reverse genetics of diplonemids in general.

Název v anglickém jazyce

Targeted integration by homologous recombination enablesin situtagging and replacement of genes in the marine microeukaryoteDiplonema papillatum

Popis výsledku anglicky

Diplonemids are a group of highly diverse and abundant marine microeukaryotes that belong to the phylum Euglenozoa and form a sister clade to the well-studied, mostly parasitic kinetoplastids. Very little is known about the biology of diplonemids, as few species have been formally described and just one,Diplonema papillatum, has been studied to a decent extent at the molecular level. Following up on our previous results showing stable but random integration of delivered extraneous DNA, we demonstrate here homologous recombination inD. papillatum. Targeting various constructs to the intended position in the nuclear genome was successful when 5 &apos; and 3 &apos; homologous regions longer than 1 kbp were used, achieving N-terminal tagging with mCherry and gene replacement of alpha- and beta-tubulins. For more convenient genetic manipulation, we designed a modular plasmid, pDP002, which bears a protein-A tag and used it to generate and express a C-terminally tagged mitoribosomal protein. Lastly, we developed an improved transformation protocol for broader applicability across laboratories. Our robust methodology allows the replacement, integration as well as endogenous tagging ofD. papillatumgenes, thus opening the door to functional studies in this species and establishing a basic toolkit for reverse genetics of diplonemids in general.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2020

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Environmental Microbiology

ISSN

1462-2912

e-ISSN

—

Svazek periodika

22

Číslo periodika v rámci svazku

9

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

11

Strana od-do

3660-3670

Kód UT WoS článku

000552503400001

EID výsledku v databázi Scopus

2-s2.0-85088447871